Cells were seeded in 24-well plates at a density of 5 × 104 cells/well and grown in complete growth media for 6 hours. The pGL4/1285 reporter construct containing the BCRP promoter (-1285/+362) upstream of firefly luciferase, the pGL4/1285Mut construct containing a mutated CRE site in the BCRP promoter, the pGL4-Basic empty vector (negative control), and an internal control vector pRL-TK (Promega, Madison, WI) which expresses Renilla luciferase, were used for various co-transfection experiments using the X-tremeGENE HP DNA transfection reagent (Roche Diagnostics). Following transfection, the cells were cultured for an additional 48 h in complete medium, then starved overnight in SFM followed by treatment with EGF (50 ng/mL) or 8Br-cAMP (2 mM) for 6 hours. Cells were lysed in passive lysing buffer (Promega) and dual luciferase assay performed. Luminescence was quantified in a TD-20/20 luminometer (Turner Designs, Inc., Sunnyvale, CA). Results are expressed as the ratio of firefly luciferase luminescence in the BCRP promoter construct relative to the Renilla luciferase luminescence in the internal control.
Passive lysing buffer
Manufactured by Promega
Sourced in United States
Passive lysing buffer is a solution used to lyse, or break open, cells in a gentle and non-disruptive manner. It is designed to extract cellular contents, including proteins and other biomolecules, without compromising their structure or function.
Automatically generated - may contain errors
2 protocols using passive lysing buffer
1
Transcriptional Regulation of BCRP
2
Measuring NRF2 Transcriptional Activity
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We measured NRF2 transcription activity by performing Luciferase reporter gene assays: 6 × 104 A549 cells were seeded in 24-well plates. Next day cells were co-transfected with 500 ng of pGL3-8xARE (NRF2 activity) or pGLHIF1.3 (HIF activity), together with 100 ng pGL4.74 (renilla luciferase). If required, cells were co-transfected with 500 ng of GFP-NRF2, GFP-NRF2-S40A or various amounts of Flag-Keap1. Transfections were performed using ViaFect (Promega, Madison, U.S.) following the manufacturer’s instructions. Seventy-two hours later, cell lysis was done by using passive lysing buffer (Promega, Madison, U.S.). Firefly and renilla luciferase were measured using the GloMax detection system (Promega) and normalized to renilla values.
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